Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

The cell envelope of Gram-negative bacteria belonging to the Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals. Here, we present the results of a genome-wide screen for cell envelope-associated resistance and susceptibility determinants in a Burkholderia cenocepacia clinical isolate. For this purpose, we construct a high-density, randomly-barcoded transposon mutant library and expose it to 19 cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profile over a hundred functional associations and identify mediators of antibiotic susceptibility in the Bcc cell envelope. We reveal connections between β-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism. The synergy of the β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. In comparison with ceftazidime, avibactam more strongly potentiates the activity of aztreonam and meropenem in a panel of Bcc clinical isolates. Finally, we characterize in Bcc the iron and receptor-dependent activity of the siderophore-cephalosporin antibiotic, cefiderocol. Our work has implications for antibiotic target prioritization, and for using additional combinations of β-lactam/β-lactamase inhibitors that can extend the utility of current antibacterial therapies.


Supplementary
Supplementary Table 6. Whole panel of CRISPRi mutants in genes associated with iron uptake from Figure 8 and their susceptibility to cefiderocol and ceftazidime in CAMHB and M9+CAA. MIC values (µg/mL) presented are medians of at least four biological replicates in the presence of 0.5% rhamnose. Bold indicates at least 2-fold difference from the NTC. Gene names are assigned by homology, if known, or else the locus tag is given (preceded by K562_).  Supplementary Figure 5. Gene fitness profiles of K56-2 transposon mutant library exposed to cell envelope-targeting antibiotic panel. Each point represents a gene that when disrupted by a transposon, affected fitness of the mutant in the presence of an antibiotic versus the DMSO control (two-sided t-test; P < 0.05; only genes with fitness effects greater than 0.5 or less than -0.5 were considered). The points are coloured based on positive (increased fitness; purple) or negative (decreased fitness; gold) interactions between the genes and tested antibiotics. The absolute difference in fitness score for a gene in each condition versus the DMSO control is given by the size of the point. Select genes of interest are indicated and named. The total number of genes in each condition with altered fitness (increased:decreased) is shown.

Gene
38 Supplementary Figure 6. BioCyc pathway enrichment of genes affecting fitness in the presence of the antibiotic panel. Genes with fitness scores either above 0.5 or below -0.5 were submitted to BioCyc for enrichment analysis (Fisher exact two-sided test without multiple testing correction) (against K56-2 genome from GenBank accession GCA_000333155.2). Enrichments from genes with positive and negative fitness scores are given in purple and gold, respectively. The dashed line indicates P = 0.05.

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Supplementary Figure 7. GO term enrichment of genes affecting fitness in the presence of the antibiotic panel. Genes with fitness scores either above 0.5 or below -0.5 were used for GO term enrichment relative to the whole K56-2 genome (two-sided hypergeometric test without multiple testing correction) with GeneMerge 1.5 (Castillo-Davis et al. 2003). Enrichments from genes with positive and negative fitness scores are given in purple and gold, respectively. The dashed line indicates equal proportions in the genome and antibiotic condition. * P < 0.05, ** P < 0.01, *** P < 0.001. Figure 9. Rhamnose dose-responses of select CRISPRi mutants constructed in this study. Cultures were grown with the indicated concentrations of rhamnose in A) LB, B) CAMHB, C) M9+CAA for 16 hours, the time at which the control strains reached the maximum OD600, and then OD600 values were recorded. Each gene was targeted with two sgRNA, and one representative is shown here. If the targeted gene does not have a 4-letter name, the locus tag (preceded by K562_) is given. NTC = non-targeting control sgRNA. Values shown are averages of at least three biological replicates (n ≥ 3); SD values are omitted for clarity.
Supplementary Figure 11. Effect of deletion and complementation of hldD and waaL on Oantigen expression. These genes were deleted in the K56-2::dCas9 background. Silver-stained SDS polyacrylamide gels of LPS extracts from mid-exponential phase deletion and complementation mutants, grown for 4 hours with or without 0.05% rhamnose. Shown are different lanes from the same gel and representative blots from two biological replicates.
Supplementary Figure 12. Concentration-and combination-dependent chemical-genetic interactions with AVI/CAZ. Comparison of gene fitness scores between A) AVI/CAZ to AVI-L, B) AVI/CAZ to CAZ-L, C) AVI-H to AVI-L, and D) CAZ-H to CAZ-L. All genes with significantly different fitness scores greater than 0.5 or less than -0.5 relative to the DMSO control (P < 0.05, two-sided t-test) were used for comparison. For the genes that were common to both conditions, the scatter plots show a Pearson's correlation test (with 95% confidence interval in light grey; two-sided) and associated r and P-values. Each point shows the average gene fitness in a given condition from three biological replicates. In the Venn diagrams, fitness scores next to genes are relative to the DMSO control.